Human immunodeficiency virus (HIV) infection has been shown by several laboratories to use a complex set of RNA synthesis and processing events in order to control viral expression. In order to determine if messages exist that have not been previously characterized, we have begun to directly analyze cDNA copies of HIV messages by Southern hybridization and DNA sequence analysis. A cDNA library in lambda gt10 has been prepared from RNA isolated from HIV-infected H9 cells. A large number of phages from this library were found to hybridize to viral sequences. A total of 19 such phages were plaque-purified an' analyzed by Southern hybridization using labeled oligonucleotides or restriction fragments derived from various locations on the HIV genome. The specific region include: (A) 5'-end of tat: (B) 5'-end of trs; (C) 5'-portion of env (excluding tat and trs region); (D) pol/sor region. Clones with the following hybridization pattern have been isolated: class I, A+B-C-D+; class II, A-B-C+D+; class III, A-B-C- D+. We have purified several of these phages and have subcloned their insert DNA into pIBI vectors. Several of these phages have been characterized by Southern blot hybridization. We are currently sequencing these DNA segments to determine if they represent novel HIV messages and, thus, might reveal new exons or splice sites.